EP3569608A1 - Composé oligosaccharidiquee pour inhiber un complexe enzymatique du facteur x de coagulation endogène, son procédé de préparation et ses utilisations - Google Patents

Composé oligosaccharidiquee pour inhiber un complexe enzymatique du facteur x de coagulation endogène, son procédé de préparation et ses utilisations Download PDF

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EP3569608A1
EP3569608A1 EP17891432.1A EP17891432A EP3569608A1 EP 3569608 A1 EP3569608 A1 EP 3569608A1 EP 17891432 A EP17891432 A EP 17891432A EP 3569608 A1 EP3569608 A1 EP 3569608A1
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oligosaccharide
formula
compound
mixture
group
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EP3569608A4 (fr
Inventor
Jinhua Zhao
Zhenguo Li
Na Gao
Mingyi Wu
Yanming Chen
Longyan ZHAO
Yongsheng Wu
Zi Li
Chuang XIAO
Shunliang ZHENG
Zhiyuan NAN
Jianbo Zhou
Jianping Xu
Lutan ZHOU
Yafang GUO
Hongbo Qin
Jikai Liu
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Mudanjiang Youbo Pharmaceutical Co Ltd
Jiuzhitang Co Ltd
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Mudanjiang Youbo Pharmaceutical Co Ltd
Jiuzhitang Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof

Definitions

  • the present invention belongs to the technical field of medicine, and in particular relates to a purified oligosaccharide compound having antithrombotic activity or a mixture of homologous compounds thereof, and a pharmaceutically acceptable salt thereof, a preparation method and uses thereof.
  • Thromboembolic diseases including ischemic stroke, coronary heart disease, venous thromboembolism are the major lethal causes of human beings.
  • Anti-thrombotic drugs such as fibrinolytic, anticoagulant and antiplatelet drugs are the basic means for clinical drug prevention and treatment of thrombotic diseases, but existing antithrombotic drugs have common defects: bleeding tendency and serious bleeding risk. Reducing bleeding tendency and bleeding risk is the core goal of the development of new antithrombotic drugs.
  • Researches in recent years have found that intrinsic coagulation pathways are closely related to pathological thrombosis, and may not be necessary for hemostasis. Therefore, intrinsic coagulation factor inhibitors have become the focus of research on antithrombotic drugs with low bleeding tendency.
  • the intrinsic factor tenase complex (Xase) is the final and rate-limiting enzyme of the intrinsic coagulation pathway, and its selective inhibitor has important potential clinical application value.
  • Fucosylated glycosaminoglycan is a glycosaminoglycan with unique chemical structures and pharmacological activities found up to now exclusively in echinoderms, which has a chondroitin sulfate-like backbone, and sulphated fucosyl (Fuc)- substituted side chains ( Yoshida et. al, Tetrahedron Lett, 1992, 33: 4959-62 ; Mour ⁇ o et. al, J Biol Chem, 1996, 271: 23973-84 ).
  • native FG has potent anticoagulant activity, and its anticoagulant mechanism is mainly related to inhibition of intrinsic Xase activity ( Thromb Haemost, 2008, 100: 420-8 ; J. Biol. Chem., 1996, 271: 23973-84 ).
  • native FG has extensive and contradictory pharmacological effects, including induction of platelet aggregation and induced decrease in circulating platelet count, activation of XII and such a side effect may cause hypotension, and so on ( Thromb Haemost, 1988, 59: 432-4 ; Thromb Haemost, 2010, 103: 994-1004 ).
  • the application value of FG under systemic administration is limited.
  • Properly depolymerized FG can reduce the activity of induction of platelet aggregation ( Thromb Haemost, 1991, 65: 369-73 ), thereby increasing its selectivity for inhibition of the intrinsic factor Xase.
  • the present inventors have previously systematically studied the chemical depolymerization of native FG and the chemical and pharmacological properties of the depolymerized product.
  • the Chinese invention patent CN 101724086 B discloses a peroxidative depolymerization method of FG, and the obtained depolymerized product can inhibit thrombus formation and the bleeding tendency is significantly reduced, however, the product obtained by the method is difficult to be further isolated and purified due to the complicated terminal structure.
  • the native FG obtained by extracting from the body wall of echinoderms by a conventional method usually also contains glucan, fucan and/or hexosamine-containing polysaccharide compounds, which have a molecular weight distribution like that of native FG.
  • These polysaccharides in native FG are difficult to be removed completely by gel chromatography, ultrafiltration or even ion exchange chromatography.
  • a comparative study conducted by the present inventors has shown that using the technical methods described in the above literatures, the natural FG extracted from the echinoderms usually contains other polysaccharide compositions in a mass ratio of about 10% ⁇ 20%. Since all of these polysaccharide compositions could be depolymerized by peroxidation, the oligosaccharide compositions of the peroxidative depolymerization product of natural FG are quite complicated.
  • Chinese invention patent CN 103214591A discloses a deacetylation-deaminative depolymerization method of FG, which can selectively cleave D-acetylgalactosamine (GalNAc)-( ⁇ 1 ⁇ 4)-D-glucuronic acid (D-GlcA) glycosidic bond, obtaining a depolymerized product containing 2,5-anhydro-D-talose (anTal) at the reducing terminal.
  • the oligosaccharide homologues in the obtained depolymerized product are composed of 3 m monosaccharide residues ( m is a natural number, hereinafter the same).
  • the deacetylation -deaminative depolymerization method cannot depolymerize glucan and fucan mixed in natural FG, and after the deacylative-deaminative depolymerization treatment, these undepolymerized polysaccharide impurities can be easily removed by gel chromatography or ultrafiltration method.
  • the deacylative-deaminative depolymerization product of natural FG has more regular structural features, and the oligosaccharide contained therein may be further isolated and purified ( CN 104370980A ).
  • nonasaccharide is the the minimum fragment with potent inhibitory activity against Xase ( Proc Natl Acad Sci USA. 2015; 112(27): 8284-9 ).
  • the present inventors' granted patent CN 201310099800 discloses a ⁇ -eliminative depolymerization method of native FG.
  • the method comprises treating FG carboxylate with a base in a non-aqueous solvent to selectively cleave D-GalNAc-( ⁇ 1 ⁇ 4)-D-GlcA glycosidic bond, thereby obtaining a depolymerized product containing unsaturated ⁇ 4,5 -hexuronic acid group ( ⁇ UA) at the non-reducing terminal, and the depolymerized product is a mixture of a series of oligosaccharide compounds.
  • the ⁇ -elimination depolymerization method since the ⁇ -elimination depolymerization method has an excellentglycosidic bond selectivity, it cannot depolymerize other types of polysaccharides contained in the native FG extract, thereby facilitating the removal of non-FG polysaccharide impurities from the FG extract.
  • the non-reducing terminals of the ⁇ -elimination depolymerization product described in CN 201310099800 are relatively regular, its reducing terminal compositions are relatively complicated: the reducing terminal residues include both "-D-GalNAc" and L-Fuc-( ⁇ 1 ⁇ 3) substituted "-4-D-GlcA". Since the structure of the reducing terminal is relatively complicated, it is technically difficult to isolate and obtain the purified oligosaccharide from the depolymerized product, and therefore it is generally preferred to directly use the depolymerized product in the form of a mixture.
  • the purified oligosaccharide has a pure chemical structure and a higher quality control level, and thus may have higher application value.
  • the degree of regularity of the terminal structure may significantly affect the technical feasibility of preparation of purified oligosaccharide compounds.
  • the ⁇ -eliminative depolymerization method can selectively cleave the "-D-GalNAc-( ⁇ 1 ⁇ 4)-D-GlcA-" glycosidic bond, and the reducing terminal of the resulting depolymerized product should be "-D -GalNAc" residue, and the oligosaccharide homologue in the resulting depolymerized product should generally be composed of 3 m monosaccharide residues.
  • a certain amount of oligosaccharide compound having "-[L-Fuc-( ⁇ 1 ⁇ 3)]-D-GlcA-" at the reducing terminal is present in the ⁇ -elimination depolymerization product of natural FG disclosed in the patent application CN 201310099800 , which indicates that during the ⁇ -elimination reaction under such conditions, there should be some side reactions, and in particular, the residue at the reducing terminal of the depolymerized product is damaged to some extent.
  • the oligosaccharide compound having -D-GlcA at the reducing terminal may account for about 10% ⁇ 30% of the total amount of the oligosaccharide compounds, and the result is similar to that of the depolymerized product of natural FG containing hemiacetal structure at the reducing terminal under the same conditions.
  • the reducing terminal of the obtained product is substantially acetylaminogalactitol group (-3-D-GalNAc-ol); while the content of the oligosaccharide compound having "-3-D-GlcA (-ol)" residue at the reducing terminal is significantly reduced, and the content may be less than about 5% or even lower than the HPGPC detection limit, according to the HPGPC area normalization method.
  • a reducing agent for example, sodium borohydride
  • the homologous oligosaccharide compounds in the resulting depolymerized product may have a more regular terminal chemical structural feature: all the homologous oligosaccharide compounds are composed of 3m monosaccharide residues; the glycosyl at the non-reducing terminal is "L-Fuc-( ⁇ 1-3)- ⁇ UA-1-" and the glycosyl group at the reducing terminal is "-3-D-GalNAc-ol".
  • the inventors By the ⁇ -elimination reaction in a basic non-aqueous organic solvent in the presence of a reducing agent and chromatographic separation technique, the inventors first isolated and purified a series of purified oligosaccharide compounds with novel chemical structures from the ⁇ -elimination depolymerization product of FG.
  • the purified oligosaccharide compounds have a common chemical structural feature: the purified oligosaccharides are composed of 3m monosaccharide residues, and the non-reducing terminal structure is "L-Fuc-( ⁇ 1-3)- ⁇ UA-1-", and the reducing terminal glycosyl group is "-3-D-GalNAc-ol".
  • an oligosaccharide containing 3m monosaccharide residues can lose a monosaccharide residue through a "peeling reaction” at the reducing terminal, thereby producing an oligosaccharide "containing (3m-1) monosaccharide residues", and the reducing terminals of such oligosaccharides are all "-D-GlcA".
  • the FG oligosaccharide containing 3m monosaccharide residues and having "-3-D-GalNAc" at the reducing end is highly susceptible to the "peeling reaction” and lose the terminal "-D-GalNAc” glycosyl group.
  • the oligosaccharide compound (which contains (3m-1) monosaccharide residues) having "-D-GlcA" at the reducing terminal produced by the "peeling reaction” is "unexpectedly” difficult to have a further “peeling reaction”.
  • the terminal "-3-D-GalNAc" glycosyl group of the depolymerized product can be further removed by the "peeling reaction" of the reducing terminal, thereby obtaining the oligosaccharide homologues with novel and regular chemical structural features.
  • HPGPC chromatographic analysis and NMR structural analysis show that in an anhydrous organic solvent, treating FG carboxyl ester with a strong base causes " ⁇ -elimination depolymerization", and then adding a small amount of strong basic aqueous solution to the reaction solution to further subject the depolymerized product of the ⁇ -elimination depolymerization to "peeling reaction", and the homologous oligosaccharide compounds contained in the depolymerized product may have a very regular chemical structure, that is, the homologous oligosaccharide compounds are composed of (3m-1) monosaccharide residues; the non-reducing terminal glycosyl group of the homologous oligosaccharide compounds is "L-Fuc-( ⁇ 1-3)- ⁇ UA-1-", and the reducing terminal glycosyl group is "-4-D-GlcA" substituted by L-Fuc at the C3 position.
  • the oligosaccharide homologue obtained by the " ⁇ -elimination” and “peeling reaction” treatment of natural FG carboxyl ester have more regular chemical structural features, and thus is easily isolated and purified to obtain a series of purified oligosaccharide compounds.
  • the common structural feature of the series of purified oligosaccharide compounds is that all the oligosaccharide compounds contain (3m-1) monosaccharide residues; the non-reducing terminal is "L-Fuc-( ⁇ 1-3)- ⁇ UA -1-"; and the reducing terminal is "-4-[Fuc-( ⁇ 1-3)]-D-GlcA".
  • the present invention can obtain a depolymerized product of FG having a more regular structure (especially a reducing terminal glycosyl structure): one is a depolymerized product having"-D-GalNAc-ol" at the reducing terminal, and the other is a depolymerized product having "-D-GlcA" at the reducing terminal. Since the terminal structure of the depolymerized product is more regular, the present invention first discloses a series of purified oligosaccharide compound derived from natural FG, which is isolated from such depolymerized products. The present invention further discloses various series of derivatives of the FG oligosaccharide compounds by structural modifications of specific chemical groups of such purified oligosaccharides.
  • the mininum structural fragment with potent inhibition against intrinsic factor Xase is nonasaccharide (NSac); for the FG oligosaccharide homologue with a reducing terminal of "-D-GlcA" and containing (3 m -1) monosaccharide residues, the mininum structural fragment with potent inhibition against intrinsic factor Xase is octasaccharide (OSac); all the purified oligosaccharide compounds also have different intensity of HC-II dependent antithrombin activity and in vitro anticoagulant activity, and have pharmacological activity of inhibiting arteriovenous thrombosis in pathological models of experimental animals.
  • the purified oligosaccharide compounds of the present invention and the oligosaccharide derivatives obtained by the structural modification thereof have coagulation factor inhibitory activity as well as significant anticoagulant and antithrombotic activity, these oligosaccharide compounds have potential application value of prevention and/or treatment for thrombotic diseases.
  • the present invention first discloses a technical method of obtaining natural FG depolymerized product with more regular chemical structure by " ⁇ -elimination depolymerization” or “ ⁇ -elimination depolymerization and terminal peeling reaction” and a FG oligosaccharide homologue with a homogenous structure obtained by such method.
  • the present invention also first discloses a purified FG oligosaccharide compound having unsaturated hexuronic acid residue structure at the non-reducing terminal, a structurally modified derivative thereof, and a mixture thereof.
  • the present invention also discloses the use of the oligosaccharide compound and a mixture thereof for the preparation of a medicament for the prevention and/or treatment of thrombotic diseases.
  • the present invention first provides an oligosaccharide compound having antithrombotic activity, particularly an activity of inhibiting intrinsic coagulation factor Xase, and a pharmaceutically acceptable salt thereof.
  • the oligosaccharide compound has a general structure represented by Formula (I): in Formula (I),
  • the oligosaccharide compound having the general structure represented by Formula (I) means a "purified oligosaccharide compound".
  • the "purified oligosaccharide compound” has a purity of no less than 95%.
  • HPGPC analytical high-performance gel chromatography
  • RID universal differential detector
  • a preferred oligosaccharide compound is the compound in which R 8 is a group represented by Formula (II), that is, the oligosaccharide compound has the general structure represented by Formula (V): in Formula (V),
  • oligosaccharide compound of Formula (I) of the present invention another preferred oligosaccharide compound is the compound in which R 8 is a group represented by Formula (III), that is, the oligosaccharide compound has the general structure represented by Formula (VI): in Formula (VI),
  • R 1 -H ;
  • oligosaccharide compound of Formula (I) of the present invention another preferred oligosaccharide compound is the compound in which R 8 is a group represented by Formula (VII), that is, the oligosaccharide compound has the general structure represented by Formula (VII): in Formula (VII),
  • R 1 -H ;
  • oligosaccharide compounds of the structure represented by the above Formula (I), (V), (VI) or (VII) of the present invention preferred oligosaccharide compounds are those in which n isoptionally 1, 2, 3 or 4.
  • the purified oligosaccharide compound of the present invention has sulfate substituents and/or free carboxyl groups, and thus can be combined with a pharmaceutically acceptable inorganic and/or organic ion to form a salt.
  • the pharmaceutically acceptable salt of the oligosaccharide compound of the present invention may be optionally an alkali metal salt, an alkaline earth metal salt or an organic ammonium salt.
  • Preferred pharmaceutically acceptable salt of the oligosaccharide compound of the present invention is a sodium salt, a potassium salt or a calcium salt.
  • the purified oligosaccharide compounds of the present invention in the form of homologues such as homologues of the compound of Formula (V), or homologues of the compound of Formula (VI) or homologues of the compound of Formula (VII) described above, are mixed, a mixture of homologous oligosaccharide compounds having a specific structure type may be obtained.
  • the present invention may also obtain a FG oligosaccharide mixture in the form of homologues having more regular chemical structure (especially a reducing terminal glycosyl structure type) by a specific technical method.
  • the present invention also provides an oligosaccharide mixture having antithrombotic activity, particularly an activity of inhibiting intrinsic factor tenase, and a pharmaceutically acceptable salt thereof.
  • the oligosaccharide mixture is composed of homologues of the above oligosaccharide compound of Formula (I); and R 8 of the oligosaccharide compound of Formula (I) in the oligosaccharide mixture is a group represented by Formula (II), or a group represented by (III), or a group represented by Formula (IV).
  • the oligosaccharide compound in which R 8 is the group represented by Formula (II) accounts for not less than 95% in the mixture, or the oligosaccharide compound in which R 8 is the group represented by Formula (III) accounts for not less than 95% in the mixture; or the oligosaccharide compound in which R 8 is the group represented by Formula (IV) accounts for not less than 95% in the mixture.
  • a preferred oligosaccharide mixture is a mixture of homologous oligosaccharide compounds having the general structure represented by the above Formula (V).
  • another preferred oligosaccharide mixture is a mixture of homologous oligosaccharide compounds having the general structure represented by Formula (VI).
  • R 1 -H ;
  • R 2 -H.
  • another preferred oligosaccharide mixture is a mixture of homologous oligosaccharide compounds having the general structure represented by Formula (VII).
  • the present invention still further provides a method for preparing the compound and the mixture.
  • the present invention provides a preparation method of the oligosaccharide compound of the structure represented by Formula (I) and a pharmaceutically acceptable salt thereof.
  • FG fucosylated glycosaminoglycan
  • the mixture of homologous oligosaccharide compounds obtained by the " ⁇ -elimination depolymerization and terminal reduction” or “ ⁇ -elimination depolymerization and peeling reaction” is isolated and purified and optionally structurally modified to obtain the desired purified oligosaccharide compound.
  • the method for preparing an oligosaccharide compound of the present invention is that for preparing an oligosaccharide compound having the structure represented by Formula (I) and having R 8 as the group represented by the above Formula (II).
  • the named "oligosaccharide compound having the structure represented by Formula (I) and having R 8 as the group represented by Formula (II)" is substantially equivalent to the oligosaccharide compound of the structure represented by Formula (V) defined above.
  • the preparation method of the oligosaccharide compound of the structure represented by Formula (V) is a " ⁇ -elimination depolymerization + terminal reduction” method.
  • the method comprises: in the presence of a strong base and a reducing agent in an anhydrous organic solvent, subjecting the carboxylated FG to a " ⁇ -elimination reaction” to cleave its "D-GalNAc-( ⁇ 1 ⁇ 4)-GlcA" glycosidic bond, and reducing the reducing terminal D-GalNAc of the depolymerized product with a reducing agent to -D-GalNAc-ol, thereby obtaining a mixture of homologous oligosaccharide compounds with relatively regular terminal structure, followed by isolating and purifying, and optional structural modifying the specific substituent to obtain the desired purified oligosaccharide compound.
  • the specific steps comprise:
  • natural FG can be understood as a polysaccharide compound formed by sequential linkage of the"trisaccharide structural units" ⁇ -4)-[L-FucS-( ⁇ 1-3)]-D-GlcA-( ⁇ 1-3)-D-GalNAcS-( ⁇ 1- ⁇ (wherein, FucS and GalNAcS represent sulfated Fuc and sulfated GalNAc, respectively).
  • natural FG typically contains a mean of about 40 to 80 of such trisaccharide structural units (approximately, the mean value of x is in the range of about 40 ⁇ 80).
  • the form in which the natural FG salt is present depends on the route of its extraction and purification.
  • the FG is present in the form of an alkali metal or alkaline earth metal salt (such as a sodium salt, a potassium salt or a calcium salt thereof).
  • an alkali metal or alkaline earth metal salt such as a sodium salt, a potassium salt or a calcium salt thereof.
  • natural FG is converted into a quaternary ammonium salt form in the step (a) ( 2 in Scheme 1).
  • the conversion of natural FG into a quaternary ammonium salt can optionally be carried out using techniques well known in the art.
  • the conversion into quaternary ammonium salt can be performed by quaternary ammonium salt precipitation method, which comprises adding an excess of an organic ammonium salt compound to an aqueous solution of an alkali metal or alkaline earth metal salt of FG, thereby forming a water-insoluble FG quaternary ammonium salt that can be easily precipitated from the aqueous solution; in addition, an alkali metal salt or an alkaline earth metal salt of FG can also be exchanged into an H-form FG using an ion exchange resin, followed by neutralization of the H-form FG with a basic organic ammonium to obtain a FG quaternary ammonium salt.
  • the step (a2) comprises convering all or part of carboxyl groups on the D-GlcA residue in the FG quaternary ammonium salt ( 2 ) into a carboxylate (3).
  • the purpose of the carboxyl esterification reaction of FG is to make it susceptible to the ⁇ -elimination reaction.
  • the GlcA in the form of a carboxyl group is less likely to undergo a ⁇ -elimination depolymerization reaction, and its carboxylate is susceptible to the ⁇ -elimination reaction due to the electronic effect of the ester group.
  • the carboxyl esterification reaction of GlcA in the FG comprises: in an organic solvent such as dimethylformamide (DMF) or a mixed solvent of DMF and a lower alcohol, a ketone and/or an ether, reacting the carboxyl group on GlcA in the FG with a stoichiometric amount of a halogenated hydrocarbon, to easily obtain a desired FG carboxylate with different degrees of esterification.
  • an organic solvent such as dimethylformamide (DMF) or a mixed solvent of DMF and a lower alcohol, a ketone and/or an ether
  • the degree of esterification of the FG carboxylate means the ratio of the number of moles of the carboxylate group formed after the esterification reaction to the number of moles of the free carboxyl group before the esterification reaction;
  • the halogenated hydrocarbon may optionally be and is not limited to: a C1-C6 linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic hydrocarbon group; or a substituted or unsubstituted C7-C12 aromatic hydrocarbon group and so on.
  • the present applicant discloses a method for the preparation of a FG carboxylate derivative in another invention patent application CN 201110318704.X , which is incorporated herein by reference in its entirety.
  • the step (b) shown in Scheme 1 comprises subjecting the FG carboxylate to ⁇ -elimination depolymerization (b1) to obtain the depolymerized product 4, and reducing the reducing terminal D-GalNAc of the depolymerized product 4 by a reducing agent to -D-GalNAc-ol (b2), and to obtain the depolymerized product 5.
  • CN 201310099800 discloses a ⁇ -elimination depolymerization method of natural FG.
  • the method can obtain a depolymerized product having unsaturated ⁇ UA at the non-reducing terminal, but the structural type at the reducing terminal of the depolymerized product is relatively complicated: the glycosyl group at the reducing terminal includes both "-D-GalNAc" and L-Fuc substituted "-D-GlcA". Since the structure of the reducing terminal is complicated, it is difficult to isolate and obtain a purified oligosaccharide from the depolymerized product, and therefore it is generally preferred to directly use the depolymerized product in the form of a mixture.
  • the ⁇ -elimination depolymerization method can selectively cleave the "D-GalNAc-( ⁇ 1 ⁇ 4)-D-GlcA" glycosidic bond, and the resulting depolymerized product has a "-D-GalNAc” residue at the reducing terminal.
  • Some amount of oligosaccharide compound having "-D-GlcA" at the reducing terminal is present in the depolymerized product of natural FG prepared by the ⁇ -elimination depolymerization method described in CN 201310099800 , indicating that there are still some side reactions under the reaction conditions. In particular, the glycosyl group at the reducing terminal of some amount of depolymerized product is destroyed.
  • the patent application CN 201310099800 is incorporated herein by reference in its entirety.
  • the purified oligosaccharide has a pure structure and a higher level of quality control, and thus may have higher application value.
  • the glycosyl group at the reducing terminal of the natural FG is reduced to an alditol by a reducing agent such as sodium borohydride, and the carboxylated product can undergo a ⁇ -elimination reaction in a basic non-aqueous solvent, but according to the HPGPC spectrum analysis of the depolymerized product, the oligosaccharide compound having -D-GlcA at the reducing terminal may account for about 10% ⁇ 30% of the total amount of the oligosaccharide compound (area normalization method), and the result is similar to that of the depolymerized product of natural FG having unreduced reducing terminal under the same conditions. This result indicates that the terminal reduction does not affect the progress of the ⁇ -elimination reaction of the FG carboxylate.
  • a reducing agent such as sodium borohydride
  • the reducing terminal of the obtained product is substantially -3-D-GalNAc-ol, while the content of the oligosaccharide compound having -3-D-GlcA-ol at the reducing terminal is very small (the content may be less than about 5%, even below the HPGPC detection limit).
  • the terminal glycosyl group of the depolymerized product obtained from the ⁇ -elimination depolymerization can be rapidly reduced to an alditol.
  • a reducing agent such as sodium borohydride
  • the reduction of the reducing terminal glycosyl group to the alditol does not affect the further ⁇ -elimination reaction of the hexuronic acid ester; on the other hand, after the terminal glycosyl group in the depolymerized product is reduced to the alditol, the destruction and degradation of the reducing terminal glycosyl group under basic conditions can be effectively avoided. Therefore, the ⁇ -elimination depolymerization of the FG carboxylate in the presence of a reducing agent can obtain a depolymerized product with a more regular chemical structure.
  • the "more regular chemical structure” means that: (1) the homologous oligosaccharide compound contained in the depolymerized product is composed of 3 m monosaccharide residues; (2) the non-reducing terminal glycosyl group of the homologous oligosaccharide compound is "L-Fuc-( ⁇ 1-3)- ⁇ UA-1-", and the reducing terminal glycosyl group is "-3-D-GalNAc-ol".
  • the technical feature of the step (b) is that the ⁇ -elimination depolymerization reaction is carried out in the presence of a reducing agent, and the ⁇ -elimination reaction condition means that the FG carboxylate is treated in a non-aqueous solvent with a strong base. Since the reaction solution for the FG carboxylic acid esterification in the step (a) is a non-aqueous solvent, after the carboxylic acid esterification reaction is completed, the reaction solution will be directly used for the ⁇ -elimination depolymerization reaction of the step (b) without further treatment.
  • the reducing agents are those that can reduce the reducing terminal glycosyl group to an alditol, such as sodium borohydride; the amount of the reducing agent is related to the amount of the depolymerized product formed.
  • the strong base in the step (b) may be optionally a lower sodium alkoxide, a diazabicyclo ring or the like.
  • the step (c) shown in Scheme 1 comprises converting the homologous oligosaccharide mixture 5 obtained by ⁇ -elimination depolymerization of FG to an alkali metal salt, which comprises optionally adding a saturated aqueous solution of an inorganic salt (such as sodium chloride) to the reaction solution.
  • the basic hydrolysis of the carboxylate of the homologous oligosaccharide compound may be generally carried out by treatment with an aqueous solution of an inorganic base (for example, 0.05 M ⁇ 1 M NaOH or KOH), thereby obtaining a homologous oligosaccharide mixture 6 containing a free carboxyl group.
  • the step (d) shown in Scheme 1 comprises isolating and purifying the oligosaccharide mixture to obtain a series of purified oligosaccharide compounds 7.
  • the isolation and purification of the oligosaccharide compound by chromatography as described in the step (d) means that the oligosaccharide compound is purified by gel chromatography and/or ion exchange chromatography, and the gel chromatography and/or ion exchange chromatography is a method well known to those skilled in the art.
  • the gel chromatography and/or ion exchange chromatography may optionally be combined with a technical method such as ultrafiltration or salting out method to increase the efficiency of the isolation and purification.
  • the step (e) shown inScheme 1 comprises optionally subjecting the oligosaccharide compound 7 obtained in the step (d) to a further structural modification, thereby obtaining the oligosaccharide compound 8.
  • the compound 8 is a oligosaccharide compound of Formula (I) in which R 8 is a group represented by Formula (II), which is equivalent to the oligosaccharide compound represented by the above Formula (V).
  • the oligosaccharide compound 7 is subjected to quaternary ammonium salt conversion, and then reacted with a halogenated hydrocarbon in an organic solvent by a conventional method in the art, to easily obtain an oligosaccharide compound of Formula (V) in which R 6 is a C1-C6 aliphatic hydrocarbon group or a C7-C12 aryl group.
  • the deacetylated oligosaccharide compound can be reacted with an acid anhydride or Et 3 N.SO 3 to obtain an N-reacylated or resulfated oligosaccharide compound, namely, an oligosaccharide of Formula (V) in which R 7 is a C2-C5 acyl group or -SO 3 H.
  • the alcoholic hydroxyl at the C1 position of -D-GalNAc-ol at the reducing terminal of the oligosaccharide compound 7 may optionally be reacted with an alcohol compound under acidic conditions to form a terminal alkylation product.
  • R 9 is a substituted or unsubstituted C1-C6 aliphatic hydrocarbon group or a C7-C12 aryl group can be obtained by the alkylation reaction.
  • the FG quaternary ammonium salt is N,N-dimethyl-N-[2-[2-[4(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy] ethyl benzammonium salt, namely benzethonium salt;
  • the organic solvent is DMF or a DMF-ethanol mixture;
  • the carboxylate is a benzyl ester; and the "complete or partially conversion into carboxylate" means that the degree of carboxyl esterification of the mixture 3 is in the range from about 30% to about 100%.
  • the organic solvent is DMF or a DMF-ethanol mixture
  • the reducing agent is sodium borohydride
  • the strong base is sodium ethoxide
  • the conversion of the quaternary ammonium salt mixture into an alkali metal salt comprises adding a saturated aqueous solution of sodium chloride to the reaction solution to convert the obtained oligosaccharide homologue 5 into a sodium salt form;
  • the basic hydrolysis in the aqueous solution means that the carboxylate of oligosaccharide compound is hydrolyzed in NaOH aqueous solution with a concentration of 0.05 M ⁇ 1 M.
  • the chromatography includes, but is not limited to, gel chromatography and/or ion exchange chromatography;
  • the further structural modification includes, but is not limited to, carboxyl esterification of D-glucuronic acid group (GlcA) and unsaturated hexuronic acid group ( ⁇ UA) in the oligosaccharide compound; deacetylation and optional reacylation or resulfation of D-acetylgalactosamine group (D-GalNAc); alkylation of alditol at the reducing terminal (D-GalNAc-ol).
  • GlcA D-glucuronic acid group
  • ⁇ UA unsaturated hexuronic acid group
  • D-GalNAc deacetylation and optional reacylation or resulfation of D-acetylgalactosamine group
  • D-GalNAc-ol alkylation of alditol at the reducing terminal
  • the reported sulfated forms of the FG side chain L-Fuc include 2,4-disulfate (L-Fuc 2S4S ), 3,4-disulfate (L-Fuc 3S4S ), 3-sulfate (L-Fuc 3S ) and 4-sulfate (L-Fuc 4S ) and no sulfate group substitution;
  • the reported sulfated forms of D-GalNAc in the backbone include 4,6-disulfate (D-GalNAc 4S6S ), 4-sulfate (D-GalNAc 4S ), 6-sulfate (D-GalNAc 6S ) and no sulfate group substitution.
  • some natural FGs may have different sulfated forms of L-FucS and/or D-GalNAcS, while other natural FGs contain a relatively regular and single sulphated form of L-FucS and/or D-GalNAcS (refer to: Pomin VH. Mar Drugs. 2014, 12, 232-54).
  • the preparation method of the oligosaccharide compound of Formula (V) that all the steps do not affect the stability of the sulfate group on the glycosyl group, and thus the sulfated form of the obtained oligosaccharide compound depends on the sulfated form of the natural FG.
  • the type of the oligosaccharide compound in its ⁇ -elimination depolymerization product is relatively small, and the oligosaccharide compounds having the same polymerization degree have the same chemical structure, and thus the purified oligosaccharide compound of the present invention can be easily prepared.
  • the side chain fucosyl group is mainly L-Fuc 2S4S
  • R 6 , R 7 and R 9 are as defined above.
  • the side chain fucosyl group is mainly L-Fuc 3S4S
  • the hexosamine in its main chain is mainly D-GalNAc 4S6S .
  • R 6 , R 7 and R 9 are as defined above.
  • Another preparation method of the oligosaccharide compound of the present invention is a method of the preparation of an oligosaccharide compound having the structure represented by Formula (I) and having R 8 as a group represented by the above Formula (III) or (IV).
  • the named "oligosaccharide compound having the structure represented by Formula (I) and having R 8 as a group represented by the above Formula (III) or (IV)" is substantially equivalent to an oligosaccharide compound of Formula (VI) or Formula (VII) as defined above.
  • the preparation method comprises " ⁇ -elimination depolymerization + peeling reaction”: subjecting the carboxylated natural FG to ⁇ -elimination depolymerization in an organic solvent in the absence of reducing agent, followed by peeling reaction to make the FG depolymerized product lose the reducing terminal D-GalNAc residue, thereby obtaining a mixture of homologous oligosaccharide compounds having -D-GlcA at the reducing terminal.
  • the method comprise the specific steps of:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 10 , R 11 and n are defined as in Formula (I) above; x, y and -COOX are defined as in Scheme 1.
  • the step (b) shown in Scheme 2 comprises subjecting the FG carboxylate to ⁇ -elimination depolymerization (b1) to form the depolymerizated product 4 (containing a small amount of product 5 ), followed by "peeling reaction” to remove the D-GalNAc residue (b2) at the reducing terminal and obtain the depolymerized product 5.
  • both oligosaccharide compound having D-GalNAc at the reducing terminal and some amount of oligosaccharide compound having "-D-GlcA-" at the reducing terminal are present, which may be related to the destruction of the reducing terminal glycosyl group of the ⁇ -elimination depolymerization product. Since the structure of the reducing terminal is relatively complicated, it is difficult to isolate and purify an oligosaccharide from the depolymerized product.
  • the step (b) first cpmprises treating the FG carboxylate with a strong base in a non-aqueous solvent in absence of a reducing agent to cause ⁇ -elimination depolymerization (b1 of Scheme 2) to obtain the depolymerized product 4 (which may contain a small amount of oligosaccharide 5 ).
  • the reaction solvent of the FG carboxyl esterification is a non-aqueous solvent
  • the reaction solution is directly used for the ⁇ -elimination depolymerization described in the step (b) without further treatment.
  • the strong base in the step (b) may be optionally a lower sodium alkoxide, a diazabicyclo ring or the like.
  • the step (b) shown in Scheme 2 further comprises converting the depolymerized product 4 into a depolymerized product 5 by further "peeling reaction” (b2 of Scheme 2), which is performed by adding a small amount of aqueous solution of a strong base to the ⁇ -elimination reaction solution.
  • the aqueous solution of the strong base may be optionally 0.25 M ⁇ 2 M NaOH, KOH or a saturated Ca(OH) 2 aqueous solution; the "small amount" of aqueous solution of a strong base means that the aqueous solution of the strong base is equivalent to about 1/5 ⁇ 1/10 of the total volume of the reaction solution.
  • the step (c) shown in Scheme 2 comprises converting the depolymerized product 5 into an alkali metal salt and hydrolyze the carboxylate, the technical method of which is the same as the method described in Scheme 1;
  • a mixture of oligosaccharide compounds 6 is isolated and purified to obtain a series of purified oligosaccharide compounds 7.
  • the purification method refers to gel chromatography and/or ion exchange chromatography, and may optionally be combined with technical methods such as ultrafiltration, salting out method to improve the efficiency of isolation and purification.
  • step (e) comprises optionally subjecting a further structural modification to the oligosaccharide compound 7 obtained in the step (d), thereby obtaining a purified oligosaccharide compound 8 or 9.
  • the oligosaccharide compound 8 is substantially equivalent to the oligosaccharide compound of Formula (VI) described above, and the compound 9 is substantially equivalent to the oligosaccharide compound of Formula (VII) described above.
  • the oligosaccharide compound 7 is subjected to quaternary ammonium salt conversion, and followed by reaction with a halogenated hydrocarbon in an organic solvent by a conventional method in the art to obtain an oligosaccharide compound in which R 6 is a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group.
  • the acetyl group on D-GalNAc in the oligosaccharide compound 7 can be removed by a hydrazinolysis method to obtain a deacetylated oligosaccharide compound.
  • the deacetylated oligosaccharide compound can in turn be reacted with an acid anhydride or Et 3 N.SO 3 to obtain an N-reacylated or resulfated oligosaccharide compound, namely, an oligosaccharide compound of Formula (VI) or Formula (VII) in which R 7 is a C2-C5 acyl or -SO 3 H.
  • the reducing terminal -D-GlcA of the oligosaccharide compound 7 can be optionally reacted with an alcohol compound under acidic conditions to form a terminal alkylation product, thereby obtaining the compound of Formula (VI) in which R 10 is optionally a substituted or unsubstituted C1-C6 hydrocarbon group or C7-C12 aryl group.
  • the aldehyde group at the C1 position of the -D-GlcA at the reducing terminal of the oligosaccharide compound 7 can be reductively aminated in the presence of an organic amine.
  • the reaction comprises reacting an organic amine with the aldehyde group at the C1 position of the terminal glycosyl group to form a Schiff base, which is reduced to a secondary amine in the presence of a reducing agent, thereby obtaining a compound (9) of Formula (VII) in which R 11 is -NHR 12 .
  • the aldehyde group at the C1 position of the -D-GlcA at the reducing terminal of the oligosaccharide compound 7 may be optionally reduced to an alditol -D-GlcA-ol using a reducing agent such as sodium borohydride, and the -D-GlcA-ol may further optionally be reacted with an alcohol compound under acidic conditions to form a terminal alkylation product, thereby obtaining the compound of Formula (VII) ( 9 ) in which R 11 is -OR 13 , and R 13 is optionally -H, a substituted or unsubstituted C1-C6 hydrocarbon group or a C7-C12 aryl group.
  • a reducing agent such as sodium borohydride
  • oligosaccharide compound represented by Formula (VI) or Formula (VII) having various specific structures defined by the present invention may be obtained by using the above structural modification method in combination.
  • the sulfated form of the obtained oligosaccharide compound also depends on the sulfated form of the natural FG.
  • R 6 , R 7 , R 10 and R 11 are defined as above.
  • echinoderms such as Holothuria scabra, Holothuria fuscopunctata and Pearsonotheia graeffei
  • R 6 , R 7 , R 10 and R 11 are defined as above.
  • the ⁇ -elimination reaction of natural FG under the technical conditions of the present invention can also be used to prepare a mixture of FG oligosaccharide compounds with more regular chemical structure.
  • the present invention further provides a method for the preparation of the oligosaccharide mixture of the present invention and a pharmaceutically acceptable salt thereof.
  • the mixture is composed of a homologue of the oligosaccharide compound having the structure represented by Formula (I) defined in the specification, and in the homologous oligosaccharide compounds of the structure of Formula (I), R 8 is the group simultaneously represented by Formula (II), simultaneously represented by Formula (III) or simultaneously represented by Formula (IV).
  • the ratio of the oligosaccharide compound of Formula (I) in which R 8 is the group simultaneously represented by Formula (II), or simultaneously represented by Formula (III) or simultaneously represented by Formula (IV) accounts for no less than 95% in the mixture.
  • natural FG is used as the starting material, and optionally, FG carboxylate is subjected to ⁇ -elimination depolymerization and terminal "reduction reaction" in the presence of a strong base and a reducing agent to obtain a mixture of homologous oligosaccharide compounds; or FG carboxylate is subjected to " ⁇ -elimination depolymerization” and terminal “peeling reaction” in the presence of a strong base to obtain a mixture of homologous oligosaccharide compounds. Then, the oligosaccharide mixture with the desired molecular weight distribution is obtained by post-treatment and optional further substituent structure modifications.
  • one of the methods is that: FG carboxylate is subjected to ⁇ -elimination depolymerization and terminal "reduction reaction" in the presence of a strong base and a reducing agent to obtain a mixture of homologous oligosaccharide compounds; the homologous oligosaccharide compound contained in the obtained oligosaccharide mixture has the general structure represented by Formula (I) defined above, and wherein R 8 is a group represented by Formula (II) defined above.
  • the method comprises the specific steps of:
  • the natural FG prepared according to the methods in the prior art also typically contains some amount of fucan, glycogen, and hexosamine-containing polysaccharide, which have a molecular weight distribution similar to FG.
  • These polysaccharide compositions have a small change in molecular weight after being treated by the above steps (a) and (b). Therefore, in the post-treatment step described in the step (c), these polysaccharide impurities can be easily removed by ultrafiltration method, dialysis method or gel chromatography.
  • the depolymerized product obtained by ⁇ -elimination depolymerization and terminal reduction treatment may also have a broader molecular weight distribution (in the oligosaccharide mixture 6 shown in Scheme 1, n may be an integer of about 0-15). Therefore, in the post-treatment step of the step (c), ultrafiltration method, dialysis method or gel chromatography treatment may be selected to remove the oligosaccharide with a higher degree of polymerization and the small molecule compounds, thereby obtaining an oligosaccharide mixture with desired molecular weight distribution.
  • the further substituent structural modification includes, but is not limited to, carboxyl esterification of D-GlcA and unsaturated ⁇ UA in the oligosaccharide compounds; deacetylation and optional further reacylation or resulfation of D-GalNAc; hydroxy-alkylation at the C1 position of the reducing terminal D-GalNAc-ol.
  • the homologous oligosaccharide mixture in which R 8 is a group of Formula (II) according to the present invention has a more regular chemical structure.
  • the oligosaccharide compound contained in the former about 10% to 30% of the oligosaccharide compounds have D-GlcA (or a derivative thereof) at the reducing terminal, and the remaining oligosaccharide compounds have D-GalNAc (or a derivative thereof) at the reducing terminal, however, the oligosaccharide compounds contained in the oligosaccharide mixture of the present invention have D-GalNAc (or a derivative thereof) at the reducing terminal, and there is no or only a trace amount of oligosaccharide compound having D-GlcA (or a derivative thereof) at the reducing terminal.
  • another method comprises: subjecting the FG carboxylate to " ⁇ -elimination depolymerization" and terminal "peeling reaction” in the presence of a strong base to obtain a mixture of homologous oligosaccharide compounds; the homologous oligosaccharide compounds contained in the obtained oligosaccharide mixture have a general structure represented by Formula (I) as defined in the specification of the present invention, and R 8 is a group represented by Formula (III) or Formula (IV) defined above.
  • the method comprises the specific steps of:
  • the homologous oligosaccharide mixture in which R 8 is a group of Formula (III) or Formula (IV) according to the present invention has a more regular chemical structure.
  • the oligosaccharide compounds contained in the former about 10% to 30% of the oligosaccharide compounds have D-GlcA (or a derivative thereof) at the reducing terminal, and the remaining oligosaccharide compounds have D-GalNAc (or a derivative thereof) at the reducing terminal; however, the oligosaccharide compounds contained in the oligosaccharide mixture of the present invention have D-GlcA (or a derivative thereof) at the reducing terminal, and have no or only a trace amount of oligosaccharide compounds having D-GalNAc (or a derivative thereof) at the reducing terminal.
  • the natural FG derived from an echinoderma such as S. Variegatus, S. horrens and S. Monotuberculatus as a starting material
  • the mixture of homologous oligosaccharide compounds of Formula (VIII), Formula (X) and Formula (XI) described above can be prepared.
  • the natural FG derived from echinoderma such as H. Scabra, H. Fuscopunctata and P.
  • the mixture of homologous oligosaccharide compounds of Formula (IX), Formula (XII) and Formula (XIII) described above can be prepared.
  • nonasaccharide is the smallest structural fragment with potent inhibitory activity of factor Xase ( Zhao LY et al., PNAS, 2015, 112: 8284-8289 .).
  • the present inventors have conducted a structure-activity relationship study on the activity of the intrinsic factor Xase (factor Xase derived from human and experimental animals) of the purified oligosaccharide of the present invention and found that:
  • the oligosaccharide compounds of the present invention and mixtures thereof have significant anticoagulant and antithrombotic activity, and when the degree of oligosaccharide polymerization is not lower than that of octasaccharide, both the oligosaccharide compounds of the present invention and the mixture thereof are an intrinsic factor Xase inhibitor with good selectivity.
  • intrinsic coagulation pathway is closely related to pathological thrombosis, and may not be necessary for physiological hemostasis.
  • Selective intrinsic coagulation pathway inhibitors may inhibit pathological thrombosis, and bleeding tendency may be effectively reduced. Since factor Xase is the terminal and rate-limiting enzyme active site of the intrinsic coagulation pathway, intrinsic factor Xase has become a drug target for the development of anticoagulant and antithrombotic drugs with low bleeding tendency.
  • the present invention further provides a pharmaceutical composition comprising the oligosaccharide or the oligosaccharide mixture.
  • the present invention provides a pharmaceutical composition having antithrombotic activity.
  • the pharmaceutical composition comprises an effective antithrombotic dose of the oligosaccharide compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the oligosaccharide compound refers to a compound having the structure represented by Formula (I) as defined in the present invention.
  • the pharmaceutical composition of the present invention is preferably prepared into a parenteral dosage form, such as an aqueous solution for injection or a lyophilized preparation formulated as an aqueous solution for injection before use, and may also be a spray administered by the respiratory tract, or a transdermal patch, a paste or a gel for transdermal administration, and so on.
  • a parenteral dosage form such as an aqueous solution for injection or a lyophilized preparation formulated as an aqueous solution for injection before use, and may also be a spray administered by the respiratory tract, or a transdermal patch, a paste or a gel for transdermal administration, and so on.
  • the oligosaccharide compounds of the present invention generally have good water solubility and are easily formulated into aqueous solutions; since the active ingredients have low molecular weights, pathogenic microorganisms and pyrogens may be removed by ultrafiltration; the optional pharmaceutical excipients for the aqueous solution and/or lyophilized preparation may include inorganic salts such as sodium chloride, buffer salts for adjusting the osmotic pressure and/or pH of the solution, and preferably include no co-solvent and/or surfactant.
  • a pharmaceutically acceptable excipient which facilitates formulation of the preparation such as mannose may be selected.
  • the oral bioavailability of the oligosaccharide compounds is relatively limited, but the oligosaccharide compounds of the present invention (especially the oligosaccharides obtained by substituent structural modifications) may still have certain pharmacodynamic activity when administered by the gastrointestinal tract.
  • the pharmaceutical compositions of the present invention may also be formulated into gastrointestinal dosage forms well known to those skilled in the art, such as a tablet, a capsule.
  • the effective antithrombotic dose of the oligosaccharide compound and its pharmaceutically acceptable salt is related to the factors such as the dosage form, the route of administration, and the weight and physiological state of the patient.
  • the content of the oligosaccharide active ingredient is in the range of about 5 mg ⁇ 100 mg; in the unit preparation form of the preferred pharmaceutical composition, the content of the oligosaccharide as an active ingredient may be in the range of about 20 mg ⁇ 80 mg.
  • the present invention also provides a pharmaceutical composition having antithrombotic activity.
  • the pharmaceutical composition comprises a potent antithrombotic dose of the oligosaccharide mixture of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the preparation form and dosage selection associated with the administration route are similar to that of the oligosaccharide-containing pharmaceutical composition mentioned above.
  • a preferred administration route is parenteral administration, especially subcutaneous injection administration or intravenous injection administration;
  • a preferred preparation form is aqueous solution for injection or a lyophilized powder for injection;
  • the oligosaccharide as an active ingredient may be present in an amount ranging from about 20 ⁇ 100 mg.
  • the oligosaccharide compound, the oligosaccharide mixture and the pharmaceutically acceptable salt thereof of the present invention have potent anticoagulant and antithrombotic activity and may be used for the prevention and treatment of thrombotic diseases, such as thrombotic cardiovascular diseases, thrombotic cerebrovascular disease, pulmonary vein thrombosis, peripheral venous thrombosis, deep vein thrombosis, peripheral arterial thrombosis. Therefore, the present invention also provides the use of the oligosaccharide compound and/or oligosaccharide mixture and a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and/or prevention of thrombotic diseases.
  • the thrombotic diseases include, but are not limited to, venous thrombosis, arterial thrombosis and/or ischemic cardiovascular and cerebrovascular diseases.
  • the present invention further provides the use of the pharmaceutical composition comprising the oligosaccharide compound and/or oligosaccharide mixture and a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating and/or preventing thrombotic diseases.
  • thrombotic diseases include, but are not limited to, venous thrombosis, arterial thrombosis, and/or ischemic cardiovascular and cerebrovascular diseases.
  • reagents used such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride, and ethanol were all commercially available analytical reagents.
  • HsFG Natural FG (sodium salt) from Holothuria fuscopunctata ; which was prepared according to the literature method ( Zhao LY et al., PNAS, 2015, 112: 8284-8289 ), with a purity of 98% (HPGPC method), and a weight average molecular weight (Mw) of about 50 kDa.
  • the used reagents such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride, and ethanol were all commercially available analytical reagents.
  • Compounds B1 ⁇ B5 are pentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide, and heptadecasaccharide, respectively, having the chemical structural formula of:
  • reagents used such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium chloride and ethanol were all commercially available analytical reagents.
  • an alcohol corresponding to a C2-C6 linear or branched alkane or an alkene may be selected to prepare the corresponding hydroxyalkylated product A8' .
  • hendecasaccharide (B3') was prepared according to the method described in Example 2, and octasaccharide (B7') was prepared according to the method described in Example 3.
  • the reagents used such as benzethonium chloride, benzyl chloride, DMF, sodium hydroxide, sodium borohydride, sodium chloride, and ethanol are all commercially available analytical reagents.
  • Sephadex G-50 medium, 50-100 ⁇ m
  • GE Healthcare product GE Healthcare product.
  • the content of the oligosaccharide compound having -D-GalNAc 4S6S -ol at the reducing terminal structure was greater than 95%.
  • the reagents used such as benzethonium chloride, benzyl chloride, DMF, DMSO, TMSD, sodium hydroxide, sodium borohydride, sodium chloride, and ethanol were all commercially available analytical reagents.
  • Ultrafiltration membrane 0.5 m 2 ) with molecular weight cutoff of 30 kDa, 10 kDa, 3 kDa, Merk Millipore.
  • the 13 C-NMR spectrum of the oligosaccharide mixture D1 is shown in Fig. 13 .
  • the 1 H-NMR of D1 there was a strong signal at 5.685 ppm, which was from 4-position hydrogen of ⁇ UA.
  • the content of the oligosaccharide compound having -D-GlcA-ol at the reducing terminal glycosyl group in D1 was more than 95%.
  • the mixture D1 was a mixture of homologous oligosaccharide compounds having a structure of L-Fuc 3S4S -( ⁇ 1,3)-L- ⁇ U-( ⁇ 1,3)- ⁇ D-GalNAc 4S6S -( ⁇ 1,4)-[L-Fuc 3S4S -( ⁇ 1,3)]-D-GlcA-( ⁇ 1,3) ⁇ n -D-GalNAc 4S6S -( ⁇ 1,4)-[L-Fuc 3S4S -( ⁇ 1,3)]-D-GlcA-ol (n is a natural number) .
  • HPGPC analysis showed that it contained pentasaccharide, octasaccharide, hendecasaccharide, tetradecasaccharide, heptadecasaccharide, eicosasaccharide, which were 3.34%, 16.71 %, 17.03%, 17.25%, 13.78%, and 12.25%, respectively.
  • oligosaccharide compounds A1 ⁇ A8 , B1 ⁇ B11 , oligosaccharide mixtures C1 , D1 , and D2 prepared according to the method described in Examples 1 ⁇ 7.
  • Reagents coagulation-controlled plasma (047B-D024A), activated partial thromboplastin time (APTT), prothrombin time (PT) assay kits, all of which were TECO GmbH company (Germany) products; Factor VIII test kit, Heparin Cofactor II (HCII), AT-dependent anti-factor IIa detection kit, AT-dependent anti-factor Xa detection kit, thrombin (factor IIa), thrombin substrate CS01 (38), KK substrate CS31 (02) were HYPHEN BioMed company (France) products; Factor VIII (FVIII), Bayer Healthcare LLC (Germany) product; ADP, Chronolog company (USA) product; sodium citrate, chloral hydrate, natural saline, were all commercial reagents.
  • Instruments XS105 electronic balance, FE20 pH meter, METTLER TOLEDO products; HH-4 constant temperature water bath, Gongyi Yuhua company product, China; VOR76X-6 vortex oscillator, Hainan Qilin Bell product; Spectrafuge-24 D907386 centrifuge, Labnet product; MC-4000 blood coagulation instrument, TICO GmbH company (Germany) product; Microplate Reder ELx 808 microplate reader, Bio-Tek company product; Chronolog-700 platelet aggregation instrument, Chrono-log company (USA) product.
  • Xase inhibitory activity assay Detection was performed according to kit instructions and literature methods by combining the Factor VIII and Factor VIII detection kits. Specifically, to each well of a 96-well plate, 30 ⁇ L of test solution, control solution, or Tris-HCl buffer (negative control) was added, and 30 ⁇ l of FVIII (2 IU/ml), 30 ⁇ l of R2 (60 nM FIXa, containing FIIa, PC/PS, Ca 2+ ) were squentially added, mixed by shaking the plate, incubated at 37 °C for 2 min; and then 30 ⁇ L of R1 (50 nM FX, containing direct thrombin inhibitor) was added, mixed by shaking the plate, incubated at 37 °C for 1 min; and then 30 ⁇ L of R3 (FXa chromogenic substrate SXa-11, about 8.4 mM) was added.
  • FVIII 2 IU/ml
  • R2 60 nM FIX
  • the absorbance at 405 nm (OD 405 ) was detected with a microplate reader, continuously measuring for 7.5 min at a interval of 30 s.
  • the Xase activity and IC 50 value of Xase inhibition of the test sample were calculated based on the OD 405 change value.
  • AT-dependent Xa inhibitory activity assay Heparin Anti-FIIa kit was used for detection. To a 96-well plate, 30 ⁇ L of sample, control solution or Tris-HCl buffer (negative control) was added, then 30 ⁇ L of 1 IU/mL AT solution was added, mixed well and incubated at 37 °C for 1 min; and than 30 ⁇ L of 8 ⁇ g/mL FXa solution was added, mixed well and incubated at 37 °C for 1 min, then 30 ⁇ L of pre-warmed 1.25 mM Xa chromogenic substrate SXa-11 was added. OD 405 was detected by a microplate reader.
  • AT-dependent IIa inhibitory activity assay Heparin Anti-FIIa kit was used for detection. To a 96-well plate, 30 ⁇ L of sample, control solution or Tris-HCl buffer (negative control) was added, and then 30 ⁇ L of 1 IU/mL AT solution was added, mixed well by shaking the plate and incubated at 37 °C for 2 min; 30 ⁇ L of 24 IU/mL FIIa solution was added, mixed well by shaking the plate and incubated for 2 min at 37 °C, and then 30 ⁇ L of pre-warmed 1.25 mM FIIa specific chromogenic substrate CS-01 (38) was added, mixed well by shaking the plate. The OD 405 was detected by a microplate reader and the IC 50 value of FIIa inhibition of each sample was calculated.
  • HC-II-dependent IIa inhibitory activity assay 30 ⁇ L of sample, control solution or Tris-HCl buffer (negative control) was added, 30 ⁇ L of 1 ⁇ M HCII solution was added, and incubated at 37 °C for 2 min; and then 30 ⁇ L of 20 NIH/mL FIIa was added, and incubated at 37 °C for 1 min; and finally 30 ⁇ L of pre-warmed 4.5 mM FIIa chromogenic substrate CS-01 (38) was added. OD 405 was detected by a microplate reader and the IC 50 value of FIIa inhibition of each sample was calculated.
  • B is the coagulation factor activity (percentage) in the presence of the test sample
  • [I] is the concentration of the test sample
  • IC 50 is the half inhibitory concentration (concentration of the test sample required to inhibit 50% of the activity)
  • n is the Hill coefficient.
  • FXII activation activity assay To a 96-well plate was added 30 ⁇ L of series concentration sample and reference solution, respectively, and then 30 ⁇ L of human standard plasma that was diluted 4 times with a 0.02 M Tris-HCl (pH 7.4) buffer containing 0.15 M NaCl was added, and incubated at 37 °C for 2 min, and then 30 ⁇ L of 6 mM kallikrein chromogenic substrate CS-31 (02) was added, and the OD 405 value was detected by a microplate reader.
  • Platelet activation activity test Anticoagulated blood was collected from healthy volunteers to prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Chronolog-700 platelet aggregation instrument and turbidimetry were used to detect platelet-induced aggregation activity of serial concentration solutions of the test sample, which were prepared by dissolving in natural saline.
  • PRP platelet-rich plasma
  • PPP platelet-poor plasma
  • Anticoagulation and coagulation factor inhibitory activity The results are shown in Table 5.
  • the oligosaccharide compounds and the mixture thereof according to the present invention have significant prolonged APTT activity, without affecting PT and TT, indicating that they can have significant anticoagulant activity against intrinsic coagulation pathway, and have no significant effect on extrinsic coagulation.
  • the oligosaccharide compounds and the mixture thereof according to the present invention have significant inhibitory activity on factor Xase; in the presence or absence of antithrombin (AT), they have no significant effect on coagulation factors such as coagulation factors IIa, Xa, XIIa, but may have a certain intensity of heparin cofactor II (HC-II)-dependent IIa inhibitory activity.
  • HC-II heparin cofactor II
  • Example B9 According to the preparation method of Example B9, a series of derivatives B9' having a corresponding C8-C12 aromatic hydrocarbon group were obtained, and according to the preparation method of Example B11, a series of derivatives B11' having a corresponding C8-C12 aromatic hydrocarbon group were obtained. They have similar activities to B9 and B11 , respectively; have drug concentration of doubling APTT clotting time of 6.0-9 ⁇ g/mL, without affecting PT and TT; have significant selective inhibitory activity against factor Xase (IC 50 , 40-110 ng/mL), and have a certain intensity of heparin cofactor II (HC-II)-dependent IIa inhibitory activity. Table 5.
  • XII activation activity analysis Within the concentration range of not more than 100 ⁇ g/ml, all the oligosaccharide compounds and oligosaccharide mixtures have no significant XII activation activity;
  • Platelet activation activity assay Within the concentration range of not more than 50 ⁇ g/ml, all oligosaccharide compounds and oligosaccharide mixtures have no significant platelet activation activity.
  • mice SD rats, weighing 250 ⁇ 350 g, male, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., license number SCXK (Xiang) 2011-0003; New Zealand rabbits provided by Kunming Medical University, SCXK (Dian) 2011-0004, used to make rabbit brain powder infusion.
  • Rats were randomly divided into 8 groups with 8 animals in each group.
  • the experimental groups and the dose of the animals in each group were (1) natural saline (NS) control group; (2) LMWH 4.0 mg/kg group; (3) A2 2.5 mg/kg group; (4) A2 group 5.0 mg /kg; (5) A2 10 mg/kg group; (6) D1 25 mg/kg group; (7) D1 5.0 mg/kg group; (8) D1 10 mg/kg group.
  • the rats in each group were administered subcutaneously (sc.) into the back, and the administration volume was 1 mL/kg.
  • the modeling experiment was performed 1 hour after administration.
  • Rabbit brain powder infusion was prepared according to the literature method ( Thromb Haemost, 2010, 103(5): 994-1004 ), and stored at - 20 °C for use.
  • Induction of inferior vena cava thrombosis by rabbit brain powder infusion The rats were anesthetized by intraperitoneally injecting with 10% chloral hydrate (300 mg/kg), the abdominal wall was cut longitudinally along the midline of the abdomen, the viscera was removed, and the inferior vena cava and its branches were isolated. A ligature was passed through the lower margin of the left renal vein of the inferior vena cava, to ligate the inferior vena cava branches below the left renal vein. The femoral vein was injected with 2% rabbit brain powder infusion (1 mL/kg). After 20 seconds, the lower margin of the left renal vein was ligated.
  • the viscera was placed back into the abdominal cavity and covered with medical gauze (infiltrated with natural saline). After 20 minutes, the blood vessel was clamped at 2 cm below the ligature, and the blood vessel was longitudinally dissected to take out the thrombus. The length of the thrombus was measured, and the wet weight of the thrombus was weighed and then dry weight was weighed after drying at 50 °C for 24 h.
  • the SPSS software was used to organize and analyze the data, and the measurement data were expressed as mean ⁇ standard deviation (x ⁇ s).
  • Data normality in different groups was tested using One-Sample KS test, variance homogeneity was tested using Levene test. If the data conformed to the normal distribution, and the variance was uniform, the significance was judged by One-Way ANOVA, otherwise, the significance was judged by Two-Independent-Samples Test.
  • mice were randomly divided into 10 groups with 8 animals in each group.
  • the experimental groups and the dose of the animals in each group were (1) natural saline (NS) control group; (2) LMWH 4.0 mg/kg group; (2) LMWH 20 mg/kg group; (3) LMWH 100 mg/ Kg group; (4) A2 5 mg/kg group; (5) A2 25 mg/kg group; (6) A2 125 mg/kg group; (7) D1 5 mg/kg group; (8) D1 25 mg/ Kg group; (10) D1 125 mg/kg group.
  • the rats in each group were administered subcutaneously (sc.) into the back, and the dose volume was 10 mL/kg.
  • mice After 60 min of subcutaneous administration in each experimental group, the mice were placed in a mouse holder, and the tail tip was cut by 5 mm by tail-clipping method, and the mouse tail was immersed in 40 mL of purified water (37°C) in the beaker. Timing was started from the first drop of blood from the cut mouse tail, and stirring was continued. At 60 min, the beaker was placed for 60 min and then the absorbance of the solution (OD540) was detected by a UV spectrophotometer.
  • purified water 37°C
  • the SPSS software was used to organize and analyze the data, and the detected data was expressed as mean ⁇ standard deviation (x ⁇ s).
  • Data normality in different groups was tested using One-Sample KS test, variance homogeneity was tested using Levene test. If the data conformed to the normal distribution, and the variance was uniform, the significance was judged by One-Way ANOVA, otherwise, the significance was judged by Two-Independent- Samples Test.
  • NaCl commercially available, pharmaceutical grade
  • Sterile water for injection 2 mL medium borosilicate tube glass bottle for injection, Millipore Pellicon 2 ultrafiltration system (Merk Millipore); VirTis Ultra 35 EL lyophilizer.
  • 1,960 vials of qualified products of A3 lyophilized preparation were obtained, and the qualified rate of the finished product was about 98%.
  • the lyophilized cake had regular appearance; the sterility, pyrogen and insoluble particulate testing were all qualified; the moisture testing results showed that the water content was less than about 3%, and the loading testing results showed that the loading was within 95 ⁇ 115% of the planned loading.
  • Oligosaccharide mixture D1 prepared according to the method described in Example 7.
  • NaCl commercially available, pharmaceutical grade
  • sterile water for injection 2 mL medium borosilicate tube glass bottle for injection, Millipore Pellicon 2 ultrafiltration system (Merk Millipore); Lyophilizer (LYO-20 m 2 ), Shanghai Tofflon Sci &Tech Co., Ltd.
  • Loading testing The gravimetric testing was in compliance with the regulations.
  • Sterility testing An appropriate amount of this product was taken and tested according to law ( 1101, Volume IV, Chinese Pharmacopoeia Edition 2015 ). The test results showed that the batch of samples met the quality requirements of injection.
  • Pyrogen testing The product was prepared into a solution containing 3.5 mg of D1 per 1 mL, and tested according to the law ( 1142, Volume IV, Chinese Pharmacopoeia Edition 2015 ), the results showed that this batch of samples met the quality requirements of pyrogen testing for injection.

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EP17891432.1A 2017-01-10 2017-01-10 Composé oligosaccharidiquee pour inhiber un complexe enzymatique du facteur x de coagulation endogène, son procédé de préparation et ses utilisations Pending EP3569608A4 (fr)

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